The invention relates to a method for the non-destructive measurement of the vitality of biological cells, especially for non-destructive determination of apoptosis of cells. The method according to the invention furthermore allows the distinction of apoptotic cells from vital cells and necrotically damaged cells. Furthermore, the present invention relates to a method for identification of substances which influence apoptosis.
In nature, cell death occurs in two different manifestations, as necrosis and apoptosis. Necrosis is cell death caused by an unspecific physical or chemical damage. By injury of the barrier function of the plasma membrane, its semipermeability for ions (especially Ca2+, Na+, K+) is disturbed. This results in massive ion and solution influx into the cell, which causes the cell to swell and finally rupture. Thus, compartmentation of the cell is abolished and the cell content is spilled. Subsequently, this process causes inflammatory reactions in the organism. Usually, necrotic damages take place within seconds to minutes.
In contrast to this, apoptosis is a programmed cell death. Apoptosis is an organized process, which is needed during the development and maintenance of an organism to eliminate unwanted or damaged cells without harming the organism in total. It is controlled by receptors (glucocorticoid receptor, TNFR, Fas, NGFR) and usually depends on de novo protein synthesis. Cellular proteases (ICE, caspases) and endonucleases become activated. Since almost all cells of an organism can undergo apoptosis, the trigger of the apoptotic process must be under permanent and strict physiological control. From the pure phenomenological viewpoint, apoptosis can be divided into six successive stages: 1. shrinkage, 2. zeiosis (plasma membrane protuberances), 3. chromatin collapse, 4. collapse of the cellular nucleus, 5. fragmentation of the nucleus into smaller units and fragmentation of cellular DNA, 6. separation of apoptotic vesicles (Thompson, C. B., 1995, Apoptosis in the pathogenesis and treatment of disease, Science 267: 1456-1462). Throughout the entire process, the barrier function of the plasma membrane is maintained. The triggering of apoptosis occurs through both internal signals of the organism as well as through external influences, such as radiation, chemical substances or reagents. Apoptosis is a process the first signs of which may develop only hours later. In contrast to necrotic cells, apoptotic cells are recognized and removed by neighbouring cells or macrophages (Orrenius, S., 1995, Apoptosis: molecular mechanisms and implications for human disease, J. Internal Medicine 237: 529-536). Here, no inflammatory reaction occurs.
Newer studies demonstrate that a multitude of diseases is based on disturbances in the regulation of apoptosis. Diseases associated with blockage of apoptosis include forms of cancer, such as carcinomas with p53 mutations and hormone-dependent tumours; autoimmune diseases, such as rheumatoid arthritis, diabetes mellitus; and viral infections, such as infections with herpes viruses, pox viruses and adenoviruses. Diseases which may be caused by an increased rate of apoptosis include particularly AIDS; neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease; the effects of toxins, such as liver diseases from large amounts of alcohol (Thompson, C. B., 1995, Apoptosis in the pathogenesis and treatment of disease, Science 267: 1456-1462).
For this reason, a multitude of methods based on apoptosis-specific parameters have been developed for determination of apoptosis. These methods, for example, are based on the determination of the phosphatidylserine portion of the extracellular cell surface by annexin V binding, determination of hypoploid nuclei, determination of cytosolic cytochrome C or caspase concentration, or the determination of caspase activation.
Detection is performed by microscopic, fluorescence-based and biochemical procedures. Electron microscopy and phase contrast microscopy, for example, detect the morphological appearance during apoptosis, such as shrinkage, membrane protuberances, and the presence of micronuclei (S. Verhaegen 1998, Microscopical Study of Cell Death via Apoptosis, European Microscopy and Analysis). However, quantification of apoptosis in a sample by microscopy requires a high degree of judgement on the part of the investigator, and manual counting of the cells after optical evaluation, or suitable image processing software.
A further well-known method for detection of apoptosis in a sample of suspended cells requires the use of a flow cytometer. Here, fluorescence dyes staining DNA are used (K. H. Elstein and R. M. Zucker 1994, Comparison of Cellular and Nuclear Flow Cytometric Techniques for Discriminating Apoptotic Subpopulations, Experimental Cell Research 211, 322-331), or a biochemical method is employed in which the process of DNA fragmentation is visualized by incorporation of nucleotides with fluorescent labels (e.g. TUNNEL method, R. S. Douglas, A. D. Tarshis, C. H. Pletcher, P. C. Nowell and J. S. Moore, 1995, A simplified method for the coordinate examination of apoptosis and surface phenotype of murine lymphocytes, Journal of Immunological Methods 188, 219-228). Also, specific fluorescently labelled probes are used for cell surface molecules. An example of such a probe is fluorescenctly labelled annexin V (G. Koopman, C. P. M. Reutlingsperger, G. A. M. Kuijten, R. M. J. Keeshen, S. T. Pals and M. H. J. van Oers, 1994, Annexin V for Flow Cytometric Detection of Phosphatidylserine Expression on B Cells Undergoing Apoptosis, Blood 84,5, 1415-1420) which binds to phosphatidylserine on the cell surface and thus visualizes the restructuring of the plasma membrane in the early apoptotic stage. However, all flow-cytometric methods for the measurement of apoptosis require calibration of the method and the system. In addition, a high number of cells (approx. 106) is required for analysis.
Other methods for the detection of apoptosis require the destruction of all cells in a sample and the gel-electrophoretic or biochemical detection of DNA fragmentation (M. Leist, F. Gantner, I. Bohlinger, P. G. Germann, G. Tiegs and A. Wendel, 1994, Murine Hepatocyte Apoptosis Induced In Vitro and In Vivo by TNF-a Requires Transcriptional Arrest, The Journal of Immunology 153, 1778-1788). These biochemical methods are procedures with several steps in which several reagents are used. This results in a very long time of analysis of up to 6 hours. In addition, several hundred cells are needed for one analysis.
Due to the relevance of disturbed regulation of apoptosis in association with a multitude of diseases, the study of apoptosis is also a central part of the search for new pharmaceutical agents, of the evaluation of their activity against diseases as well as in environmental analytics. Generally, a compound which influences apoptosis must not only be capable of modulating apoptosis but also of permeating the intact cell membrane. Furthermore it is assumed that a multitude of cellular receptors, proteins, cell components and cofactors influences the process of apoptosis in living cells.
Therefore, a multitude of screening methods has already been developed for the identification of substances which influence apoptosis, which are based on the determination of apoptosis-specific parameters in the cell or in cell-free systems.
For example, WO 98/02579 describes a screening assay for the identification of apoptosis-regulating substances in a cell-free system. Here, several apoptosis-specific parameters, such as cytochrome c or CPP32 protease activity, are determined as measures of apoptosis. For screening, a supernatant obtained at 100,000×g from the cytosol of non-apoptotic cells is employed. The determination of substances influencing apoptosis was performed by addition of test compounds to this supernatant which may have negative, positive or no effect on apoptosis. The results obtained in this way are compared to those of a reference substance.
WO 98/55615 describes a screening assay for determination of therapeutically active substances influencing apoptosis. Cell-free assays are described which are designed to study compounds that compete with cytochrome c for binding to apaf-1. Furthermore, assays for determination of substances influencing apoptosis are also described which are based on the study of the proteolytic cleavage of caspase-3 precursors in presence or absence of potential substances influencing apoptosis.
In WO 99/18856, a method is described for the detection of substances for induction or inhibition of enzymes of the apoptosis cascade, especially caspases. The method is performed with whole cells or tissues. In order to do so, a portion of the cells is treated with the test compounds and fluorescenctly labelled reporter molecules, whereas another fraction is treated only with the reporters, as control. A change in fluorescence compared to the control sample indicates that a substance to be tested influences enzymes of the cascade.
Taken together, all currently known methods for determination of apoptosis or apoptosis-modulating substances have a multitude of disadvantages. Thus, they frequently require a great deal of time due to elaborate experimental steps and in addition require large quantities of, for example, antibodies, dyes and other reagents that can enter the cell. Furthermore, most of the methods are based on final cell lysis, eventually leading to destruction of the cells. Other investigated parameters, such as e.g. annexin V, are not specific for apoptosis, still other methods require a large number of cells. Furthermore, many methods are only suited to study late stages of apoptosis which frequently do not occur in vivo, since the apoptotic cells have already been taken up by neighbouring cells or macrophages by phagocytosis in this stage.
Frequently used cell-free assays, such as the determination of cytochrome c and other apoptosis-specific markers, are not suitable for determination of the ability of substances affecting apoptosis to pass through the intact cell membrane.
Furthermore it is though that different cell types have different receptors and cofactors for modulation of apoptosis. For this reason it is not possible to find specific or organ-specific apoptosis modulators with cell-free assays.
Due to the fact that in most of the known methods the importance of cellular receptors and other cofactors is neglected, the danger of identification of false-positive or false-negative substances influencing apoptosis is high, since these substances do not show any or do not show the expected effect in living cells. In addition, substances influencing apoptosis that modulate apoptosis indirectly through one of these receptors or cofactors are not detected.
Considering these disadvantages of the known methods, it would be desirable to have a method available that is suitable for the unambiguous identification of apoptosis and determination of substances which modulate apoptosis. This method should be able to recognize apoptosis in an early stage, and should be easily performable, even with whole cells.
Dielectrophoretic techniques, such as e.g. the dielectric single particle spectroscopy (R. Pethig and G. H. Markx, 1997, Applications of dielectrophoresis in biotechnology, Trends in Biotechnology 15, 426-432) and electrorotation (T. Schnelle, T. Müller and G. Fuhr, 1999, Dielectric single particle spectroscopy for measurement of dispersion, Medical & Biological Engineering & Computing 37, 264-271) are already known as such.
Also, devices for the measurement of electrorotation in combination with the use of optical tweezers are already known (T. Schnelle, T. Müller, C. Reichle and G. Fuhr, 2000, Combined dielectrophoretic field cages and laser tweezers for electrorotation, Applied Physics B, Lasers and Optics, Springer-Verlag) and automatic signal uptake (C. Reichle, T. Müller, T. Schnelle and G. Fuhr, 1999, Electro-rotation in octopole micro cages, Journal of Physics D: Applied Physics 32, 2128-2135; De Gasparis, Wang, Yang, Becker and Gascoyne, 1998, Meas. Sci. Technol. 9, 518-529).
The object of the present invention is to provide an improved method for non-destructive measurement of vitality, especially for the detection of apoptosis, in biological cells. The invention is intended to allow the provision of an improved method for identification of substances influencing apoptosis.